Dynein–Dynactin–NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

Okumura, M; Natsume, Toyoaki; Kanemaki, Masato T.; Kiyomitsu, Tomomi

doi:10.7554/elife.36559

Cited by 127 publications

(199 citation statements)

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“…In the presence of centrosomes, the minus-end of spindle microtubules and K-fibers are sorted into the spindle poles (Khodjakov et al, 2003;Maiato et al, 2004;Goshima et al, 2005), where centrosomes predominantly provide the site for microtubule minus-end focusing. As previously described (Okumura et al, 2018), in this situation, the clustering of NuMA is not absolutely required for spindle pole focusing. This finding was also confirmed in the present study (Appendix Fig S6C and D).…”

Section: Discussionmentioning

confidence: 57%

“…HCT116 TetOsTIR1 and HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells were provided by Dr. Masato Kanemaki and Dr. Toyoaki Natsume (Natsume et al, 2016). The HCT116 TetOsTIR1 NuMA-mAID-mClover-3X-FLAG, HCT116 TetOsTIR1 NuMA-mAID-mClover-3X-FLAG DHC1-SNAP mCherry-NuMA WT, and HCT116 TetOsTIR1 NuMA-mAID-mClover-3X-FLAG DHC1-SNAP mCherry-NuMA 5A-3 were provided by Dr. Tomomi Kiyomitsu (Okumura et al, 2018). HCT116 CMVOsTIR1 HsSAS6-AID have been described previously (Yoshiba et al, 2019).…”

Section: Methodsmentioning

confidence: 99%

“…Thus, the spindle pole is organized by NuMA and the dynein-dynactin complex (Merdes et al, 1996(Merdes et al, , 2000Hueschen et al, 2017). In addition to their functions in spindle pole organization, NuMA and the dynein-dynactin complex cooperate to regulate spindle positioning by forming dynein-dynactin-NuMA clusters at the mitotic cell cortex through the clustering activity of NuMA (Okumura et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…The clustering activity of NuMA partially contributes to NuMA assembly in acentrosomal spindle poles It has been shown that NuMA forms oligomeric structures in vitro (Harborth et al, 1999), and its clustering activity is important for the generation of spindle-pulling forces at the cortex (Okumura et al, 2018). These observations raised the question of whether the clustering activity of NuMA is also required for acentrosomal spindle pole formation.…”

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confidence: 99%

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Nu MA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells

et al. 2019

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In most animal cells, mitotic spindle formation is mediated by coordination of centrosomal and acentrosomal pathways. At the onset of mitosis, centrosomes promote spindle bipolarization. However, the mechanism through which the acentrosomal pathways facilitate the establishment of spindle bipolarity in early mitosis is not completely understood. In this study, we show the critical roles of nuclear mitotic apparatus protein (NuMA) in the generation of spindle bipolarity in acentrosomal human cells. In acentrosomal human cells, we found that small microtubule asters containing NuMA formed at the time of nuclear envelope breakdown. In addition, these asters were assembled by dynein and the clustering activity of NuMA. Subsequently, NuMA organized the radial array of microtubules, which incorporates Eg5, and thus facilitated spindle bipolarization. Importantly, in cells with centrosomes, we also found that NuMA promoted the initial step of spindle bipolarization in early mitosis. Overall, these data suggest that canonical centrosomal and NuMA-mediated acentrosomal pathways redundantly promote spindle bipolarity in human cells.

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Section: Discussionmentioning

confidence: 57%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Nu MA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells

et al. 2019

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“…In most animals and fungi, pulling forces for orienting the spindle are generated by the microtubule motor cytoplasmic dynein, and are dependent on the anchoring of the dynein motor at the cell periphery (Bowman et al, 2006; Couwenbergs et al, 2007; Du and Macara, 2004; Heil-Chapdelaine et al, 2000; Kotak et al, 2012; Kotak et al, 2014; Nguyen-Ngoc et al, 2007; Sana et al, 2018). It has been proposed by recent work that cortical dynein-anchoring proteins might enhance spindle pulling function by forming clusters on the cell membrane (Okumura et al, 2018; Seldin et al, 2016); a favored hypothesis is that clustering of anchoring proteins contributes to the generation of large cooperative pulling forces by increasing the number of interacting motors per cortical MT contact site (Kiyomitsu, 2019; Okumura et al, 2018), analogous to how lipid microdomains on phagosomes facilitate motor clustering to achieve cooperative force generation of dynein during intracellular trafficking (Rai et al, 2016). However, despite identification of the domain required for clustering activity and punctate localization in both yeast and mammalian dynein-anchoring proteins (Harborth et al, 1999; Okumura et al, 2018; Tang et al, 2012), factors influencing cluster assembly, size, and distribution along the cortex are poorly understood.…”

Section: Introductionmentioning

confidence: 99%

Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

Omer

Brock

Beckford

et al. 2020

Preprint

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13 Current model for spindle positioning requires attachment of the microtubule (MT) motor 14 cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect 15 spindle displacement. How dynein is anchored by cortical attachment machinery to generate 16 large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the 17 yeast dynein attachment molecule, is limited by Mdm36. Overexpression of Mdm36 results in an 18 overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that 19 mediate dynein-anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 20 clusters containing only ~6 copies of Num1 molecules mediate dynein-dependent spindle 21 pulling via lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with 22 mitochondrial tethering but not dynein-based spindle-pulling function of Num1. We propose that 23 formation of small ensembles of attachment molecules is sufficient for dynein anchorage and 24 cortical generation of large spindle-pulling force. 25 26 4 al. , 2010; Lackner et al., 2013). Although it is well-accepted that dynein exerts spindle pulling 53 force at cortical Num1 sites, the abundance and heterogeneity of Num1 patches along the cell 54 cortex (Heil-Chapdelaine et al., 2000; Omer et al., 2018; Schmit et al., 2018) has made it 55 impossible to follow the effects of astral MT plus end interaction with individual cortical Num1 56 sites, a prerequisite for understanding how clustering might impact dynein force amplification. 57 To our knowledge, contacts between astral MT plus end and individual Num1 foci have not 58 been observed for MT sliding, the in vivo hallmark of dynein-mediated spindle pulling (Adames 59 and Cooper, 2000; Yeh et al., 2000), hence the size of Num1 clusters required for this classic 60 dynein-dependent microtubule-cortex interaction remains unknown. Additionally, recent work61 shows that organelles such as mitochondria and endoplasmic reticulum (ER) are involved in 62 regulating Num1 cluster formation: a subset of cortical Num1 clusters appears to require 63 mitochondria for their assembly (Kraft and Lackner, 2017), whereas another population requires 64 the ER tethering proteins Scs2/Scs22 for their distribution throughout the cell cortex (Chao et 65 al., 2014; Omer et al., 2018). The general hypothesis emerging from these studies is that 66 distinct populations of Num1 clusters might exist at the cell periphery, but whether different 67 pools of Num1 could be performing different Num1 functions -namely, dynein anchoring and 68 mitochondrial tethering -remains a total mystery.69 Here, we set out to characterize the role of Mdm36 in Num1 clustering and found that, in 70 contrast to the prevailing notion for dynein-anchoring proteins, enhancing Num1 clustering 71 unexpectedly reduces dynein recruitment to the cell cortex, but without affecting dynein function 72 in spindle positioning. We report direct observation of MT sliding occurring upon...

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Expression and cell transformation activity of dynactin‐associated protein isoforms

et al. 2021

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Overexpression of human dynactin-associated protein isoform a (dynAPa) transforms NIH3T3 cells. DynAPa is a single-pass transmembrane protein with a carboxy-terminal region exposed to the outside of cells. According to the NCBI RefSeq database, there may be two other splicing variants of the encoding gene (dynAPb and c). DynAPa and c differ in some amino-terminal residues (NH 2 -MVA in dynAPa and NH 2 -MEYQLL in dynAPc). DynAPb has the same amino-terminal residues as dynAPc, but lacks 55 residues in the intracellular region. All three isoforms have the same carboxy-terminal region, including the transmembrane domain.Expression of mRNAs of three splicing variants was found in human cancer cell lines ACHN and Caki-1. The subcellular localization and in vitro cell transformation ability of the three isoforms were examined using NIH3T3 cells overexpressing each respective isoform. All isoforms were found to be localized to the Golgi apparatus and plasma membrane, where the carboxy-terminal region was exposed to the outside of cells. Cell transformation was tested using focus formation due to loss of contact inhibition of cell proliferation, and colony formation was examined on soft agar and spheroid formation in ultralow U-bottomed wells. DynAPa robustly formed foci and colonies on soft agar and spheroid, whereas these abilities were considerably decreased for dynAPb and completely lost in dynAPc. These findings warrant dissection studies to identify the dynAP domain that is required for cell transformation.

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Dynein–Dynactin–NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

Cited by 127 publications

References 59 publications

Nu MA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells

Nu MA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells

Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

Expression and cell transformation activity of dynactin‐associated protein isoforms

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